PROJECT SUMMARY Colorectal cancer (CRC) is the second leading cause of cancer-related death in the Western world, and there are no effective therapies for patients with advanced disease. Large chromosome rearrangements involving two members of the RSPO family of WNT pathway co-agonists were recently described in 5-10% of human CRC, and offer a potential target to treat this subset of patients. However, we still do not fully understand how specific RSPO fusion proteins (e.g. PTPRK-RSPO3) contribute to tumorigenesis, and whether targeting this genomic change will provide meaningful therapeutic responses and improved patient outcomes. To begin to delineate the consequences of RSPO fusions, we developed genetically engineered animal models in which endogenous chromosome rearrangements can be generated in the intestine using an inducible CRISPR/Cas9 platform we pioneered. Using this approach, we provided the first evidence that Ptprk-Rspo3 chromosome rearrangements are sufficient to initiate tumor development in the gut. Moreover, tumors derived from endogenous chromosome alterations produce phenotypes distinct from published models that induce ectopic overexpression of an Rspo3 cDNA. Thus, defining the molecular consequences of specific fusion events is likely critical for understanding the true impact of such rearrangements on tumor biology. In addition, we showed that Ptprk-Rspo3 fusion lesions growing within the native intestinal mucosa are exquisitely sensitive to drugs that block WNT secretion, but that the accumulation of genetic alterations can influence drug response. Based on this work, we will test the hypothesis that Ptprk-Rspo3 fusion proteins provide a WNT-dependent cell intrinsic growth advantage due to the endogenous fusion event, but that the accumulation of cooperating oncogenic insults promotes WNT independence and drug resistance. Using ex vivo organoid model systems, Aim 1 will determine the molecular consequences of specific RSPO fusions and delineate how specific fusion events and loss of the fusion partner (PTPRK) promote cell transformation. In Aim 2, through sequential CRISPR-based editing in organoids and orthotopic tumor models, we will define genetic landscape and molecular mechanisms linked to therapy failure and acquired drug resistance. Together, this work will contribute significantly to a molecular understanding of how RSPO genomic rearrangements impact oncogenic WNT signaling, and has the potential to make an immediate and significant clinical contribution, by defining the mechanisms that influence tumor progression and therapy resistance. We expect our studies will reveal ways to more effectively identify patient populations that would respond to treatment and/or predict combination and second line therapies for treatment refractory tumors. Thus, results from this study will be a significant step toward the overall goal of safe and effective targeted therapies for CRC, and improved patient outcomes.